Rutin As An Agonist Of PPAR-γ And Study Of Polar State Transformation Of Macrophages

Objective To study the effect of Rutin on the polarization state of macrophages,to clarify the mechanism of Rutin against atherosclerosis,and provide new support for its clinical application.Methods(1)Select and culture RAW264.7 macrophages without differentiation stimulation,and use MTS to detect cell proliferation activity at different drug concentrations.(2)Stimulate macrophages RAW264.7 to polarize.Differentiate into proinflammatory Ml macrophages through having been stimulated with LPS(200ng/mL)+IFN-γ(2.5 ng/mL)and stimulate to differentiate into anti-inflammatory M2macrophages with IL-4(10 ng/mL).The unpolarized RAW264.7 macrophages were seeded in a cell culture dish at 5×10 ~4cells/ml,and after the cells were adhered,the cells were replaced with stimulatory drugs and cultured for 24 hours.Afterwards,the polarization of the cells was successful.Immunofluorescence was used to detect the expression of iNOS,and PPAR-γexpression in each group.Western blot was used to detect the expression of CD68 and iNOS in M1 macrophage markers and the expression of CD206 and Arg-1 in M2 macrophage markers.And compare with the control group data.(3)M1 and M2 type RAW264.7 macrophages in the polarized group were aspirated from the original culture medium 24 hours after the cells were stimulated by polarized drugs,and incubated with different concentrations of Rutin culture solution.Western Blot was used to detect the expression of CD68 and iNOS in M1 macrophage markers,and the expression of CD206 and Arg-1 in M2 macrophage markers.The results were compared with those of control and model groups.Expression levels of M1 macrophage marker genes IL-6 and iNOS,and expression levels of M2 macrophage marker genes Arg-1.(4)PPAR-γinhibitors were added and real-time PCR and Western Blot were used to detect PPAR-γgene expression,the expression and secretion of inflammation-related cytokines,and M1 and M2 giants.The expression of phagocytic markers has determined that Rutin affects the specific pathway of macrophage polarization.Results(1)M1 macrophages showed higher levels of inflammatory cytokine iNOS and M1 marker CD68 than M0 cells;M2 macrophages thought to secrete more IL-10and M2 marker CD206 in M0 macrophages.Increased volume.It was shown that M1 macrophages were successfully polarized after LPS(200 ng/mL)+IFNγ(2.5 ng/mL)stimulation for 24 h,and M2 cells were successfully polarized after IL-4(10 ng/mL)stimulation for 24 h.(2)Under the action of Rutin,CD68 marker of M1 macrophage decreased,and the expression levels of related inflammatory factors IL-6 and iNOS decreased.(3)Under the action of Rutin,M2 macrophage marker CD206 was decreased.Rising.(4)Rutin is used as a PPAR-γgene activator to inhibit the expression of NF-κB gene and increase the expression of p-STAT6 gene so as to achieve the purpose of reversing M1 polarization.Conclusion(1)Rutin function as the Agonist of the PPAR-γgene.(2)Rutin can inhibit the expression of inflammatory cytokines in M1 macrophages and maintain its polarization to M2 macrophages.